NK cell analysis using CD16PE and CD56PE

I wanted to identify NK cells with CD3/CD16+56 combination AND using CD3 Pecy5 and CD16 PE and CD56 PE. So far, I have tried the latter combination using doses of CD16PE and CD56PE ranging from 2- 20ÁL/test. However, the results are very different from staining when I used the Simultest CD3FITC/CD16+56PE mix in parallel.

Use different channels for markers. CD16-FITC/CD56-PE/CD3-PE-Cy5 could be the choice for NK analysis.

P.S. How many FLs your cytometer has?

Dear Denis

I am using FACScan to measure intracellular IFN-g response from NK cells. By mistake I bought simultest CD3FITC/CD16+56PE. What I am planning to do is that using CD16PE and CD56PE from Pharmingen and try to get the optimal concentration, just like simultest result. My second option is to use another flurochrome for ifn-g (which is read by the FL3) and use the Simultest.

For more information, here is the email conversation that I had with a BD technical suppport.

My email

I wanted to identify NK cells with CD3/CD16+56 combination AND using CD3 Pecy5 and CD16 PE and CD56 PE. So far, I have tried the latter combination using doses of CD16PE and CD56PE ranging from 2- 20µL/test. However, the results are very different from staining when I used the Simultest CD3FITC/CD16+56PE mix in parallel.

Do you have experience with the detection of NK cells using the 555516 and 555407 reagents, and can you recommend appropriate doses or any changes in protocol?

BD Technical response

I have summarized the bits of information in each of our emails. I have included extra information that can help us to understand the different stainings you get.

"I wanted to identify NK cells with CD3/CD16+56 combination AND using CD3 Pecy5 and CD16 PE and CD56 PE. So far, I have tried the latter combinationusing doses of CD16PE and CD56PE ranging from 2- 20µL/test. However, the results are very different from staining using the Simultest CD3FITC/CD16+56PE mix."

So; here we have many things that are different, starting from the clones of the antibodies used in the simultest product when compared to the pharmigen product.

Next difference is much certainly the concentration; and even if the concentrations are the same, since it is not the same clone; one cannot compare.

Third; the way to purify the antibodies, removing free fluorochroms; etc is different between simultest products and pharmigen products. This would lead to a difference in background staining and intensity of real signal. For pharmigen products; it is important to use isotype controls which are also pharmigen; like this you will know exactly where to set your gates and discriminate between specific and unspecific binding.

The simultests products are CEIVD and they are quite strict in their protocols. The conc used in their mixed are proprietary to compensate for all the work to adjust the perfect titration.

I hope this information helps you to understand the differences you get in both stainings and how to solve it or analyse it.